Biotech Juni 2015 Rev 19-9-16 New.Indd

HerdiniIndonesian et al. Journal of Biotechnology, June, 2015 Vol. 20, No.I.J. 1, pp.34-41Biotech. Diversity of Nonribosomal Peptide Synthetase Genes in the Anticancer- Producing Actinomycetes Isolated from Marine Sediment in Indonesia Camelia Herdini1,5, Shinta Hartanto1, Sofi a Mubarika1, Bambang Hariwiyanto1,5, Nastiti Wijayanti1, Akira Hosoyama2, Atsushi Yamazoe2, Hideaki Nojiri3, Jaka Widada4 1 Graduate School of Biotechnology, Universitas Gadjah Mada, Barek Utara, Yogyakarta, Indonesia 2 Biological Resource Center, National Institute of Technology and Evaluation, Nishihara, Shibuya-ku, Tokyo, Japan 3 Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo, Japan 4 Department of Agricultural Microbiology, Universitas Gadjah Mada, Bulaksumur, Yogyakarta, Indonesia 5 ORL Head and Neck Department, Faculty of Medicine, Universitas Gadjah Mada, Sekip, Yogyakarta, Indonesia Abstract Marine actinomycetes is a group of bacteria that is highly potential in producing novel bioactive compound. It has unique characteristics and is different from other terrestrial ones. Extreme environmental condition is suspected to lead marine actinomycetes produce different types of bioactive compound found previously. The aim of this study was to explore the presence and diversity of NRPS genes in 14 anticancer-producing actinomycetes isolated from marine sediment in Indonesia. PCR amplifi cation and restriction fragment analysis of NRPS genes with HaeIII from 14 marine actinomycetes were done to assess the diversity of NRPS genes. Genome mining of one species of marine actinomycetes (strain GMY01) also was employed towards this goal. The result showed that NRPS gene sequence diversity in 14 marine actinomycetes could be divided into 4 groups based on NRPS gene restriction patterns. Analysis of 16S rRNA gene sequences of representatives from each group showed that all isolates belong to genus of Streptomyces. Genome mining result showed that strain GMY01 harboring 10 different NRPS gene clusters that encode secondary metabolites, as pure NRPS or hybrid between NRPS and other compounds. These results indicated that marine actinomycetes having a high potential to be developed as source of anticancer drugs development. Keyword: marine actinomycetes, non-ribosomal peptide synthetase genes diversity, Streptomyces, genome mining, anticancer drugs development Introduction actinomycetes decreased, whereas the rate of Wide range of bioactive compounds re-isolation of known compounds increased metabolites were isolated and identified (Dharmaraj, 2010). Thus, it is crucial that new from soil actinomycetes. Recently, the rate group of actinomycetes from unexplored of new metabolites discovery from terrestrial habitats to be pursued for new sources of bioactive compounds, one of which is marine actinomycetes (Magarvey et al., 2004). * Corresponding author: Nonribosomal peptide synthetases Camelia Herdini (NRPS) and type I polyketide synthases Graduate School of Biotechnology, Universitas (PKS-I) are biosynthetic systems involved in Gadjah Mada, Barek Utara, Yogyakarta, Indonesia. Email: [email protected] or Jaka Widada the synthesis of a large number of important [email protected]) biologically active compounds produced 34 Herdini et al. I.J. Biotech. by microorganisms, among others by Material and Methods actinomycetes (Ayuso-Sacido and Genilloud, Bacterial strains and culture conditions. 2004). The 14 anticancer-producing Farida et al. (2007) isolated several actinomycetes isolated from marine marine actinomycetes from Krakal Beach, sediment in Indonesia used in this study Gunung Kidul, Yogyakarta and proved the were from previously study (Farida et al., anticancer effects of the marine actinomycetes 2007). All strains used in this study were extracts. That research results also showed grown in shaking fl asks containing starch that the anticancer compounds from marine nitrate broth with fi ltered seawater at room actinomycetes suggested maybe belong to temperature. All strains were stored at -80°C the poliketide or non-ribosomal peptides as suspensions of spores and hyphae in 15 % based on the presence of NRPS or PKS gene. (v/v) glycerol. According to Wang et al. (2014), most of the screening methods performed were to explore Isolation of chromosomal DNA a new bioactive compounds that focusing on Chromosomal DNAs were isolated by the detection of NRPS or PKS genes. NRPS a versatile quick-prep method for genomic gene is one of the genes involved in the DNA of Gram-positive bacteria (Pospiech and synthesis of a variety of important bioactive Neumann, 1995), with some modifications. compounds called non-ribosomal peptides. Bacterial culture (1 ml) grown in a starch nitrate Many researchers have made isolation broth shake culture were centrifuged, rinsed process of bioactive compounds from with TE and re-suspended in 0.4 ml TE buffer. terrestrial actinomycetes, but after the Lysozyme-1 was added to a concentration of 1 elucidation process, bioactive compounds mg ml and incubated at 37°C for 1 h. Then 0.1 were often structurally similar to the structure vol 10 % SDS was added and incubated at 65°C of bioactive compounds that was found with occasional inversion for 2 h. One-third previously, so information about NRPS genes volume 5 M NaCl and 1 vol. chloroform were sequence and diversity in actinomycetes added and incubated at room temperature for are very important to avoid the risk of re- 0.5 h with frequent inversion. The mixture was isolation. This approach can be used as a tool centrifuged at 13,000 rpm for 10 min and the for initial screening in the discovery of new aqueous phase was transferred to a new tube bioactive compounds from actinomycetes. using a blunt-ended pipette tip. Chromosomal Referring to research conducted by Farida DNA was precipitated by the addition of 1 vol. et al. (2007), although marine actinomycetes 2-propanol with gentle inversion. The DNA that could potentially have anti-cancer was transferred to a new tube, rinsed with 70 % compounds and detected carry NRPS gene ethanol, dried under vacuum and dissolved in in the genome, but these studies have not a suitable volume (about 50 ml) of TE buffer. observed NRPS genes sequence and their diversity. NRPS gene PCR Amplification and The aim of this study was to explore Restriction Endonuclease Digestion the presence and diversity of NRPS genes in The NRPS gene was amplified using 14 marine actinomycetes. PCR amplifi cation primers A3 (5’GCSTACSYSATSTACA and restriction fragment analysis of NRPS CSTCSGG’3) and A7R (5’SASGTCVCCSGTS genes from different species of marine CGGTAS ‘3) (Ayuso-Sacido and Genilloud, actinomycetes were done. Genome mining 2004). Amplifi cation of the NRPS gene was of one species of marine actinomycetes (strain performed in T100 thermal cycler (Bio-Rad) GMY01) also was employed towards this in a total volume of 50 ml containing 30-50 ng goal. DNA, 100 mM each primer, 10 mM dNTP, 10X buffer and 1.5 U Taq DNA polymerase 35 Herdini et al. I.J. Biotech. (Promega). PCR program used to follow (NGS) using 454 pyrosequencing technology the procedures performed by Farida et al. (454 GS FLX) and HiSeq1000 (Illumina) (2007) with modifications. PCR program (Herdini et al., unpublished). Genome mining included initial denaturation at 97° C for 3 min, and analysis of gene cluster involved in the denaturation at 95°C for 1 min, then followed by biosynthesis of secondary metabolites of annealing at 55°C for 1 min, extension at 72°C Streptomyces sp. GMY01, especially gene for 3 min the whole process takes place as many clusters containing NRPS, were done with as 30 cycles, the last cycle followed by a fi nal antiSMASH 3.0 (Medema et al., 2011; Weber extension at 72°C for 10 min and cooling stage et al., 2015) available on http://antismash. at 4°C. The PCR product was visualized by 1% secondarymetabolites.org. agarose electrophoresis. Restriction enzyme, HaeIII, was used to digest the amplifi ed NRPS Data analysis genes fragment. The reactions were performed Data subjected from electrophoresis in fi nal volumes of 20 μl containing at least 8 were analyzed using UPGMA method. μg∙ml-1 of NRPS genes products at 37°C for 3 h. The sequences of 16S rRNA determined in The digestions were then analyzed by agarose this study were aligned using CLUSTAL gel electrophoresis (2%, w/v) for 1 h at 110 W software (Thompson et al., 1994). The V followed by visualization of the banding nucleotide similarity values were calculated patterns using a UV transilluminator (UVP from the alignment. Evolutionary trees for the Canada). datasets were inferred from the neighbour- joining method (Saitou and Nei, 1987) using PCR Amplifi cation and sequencing of 16S MEGA version 6.0 (Tamura et al., 2013). The rRNA genes stability of relationships was assessed by The 16S rRNA gene was amplified performing bootstrap analysis of neighbour- using primers 27F (5’-AGA GTT TGA TCC joining data based on 1,000 resampling. TGG CTC AG-3 ‘) and1492 R (5’-GTT TAC CTT GTT ACG ACT T-3 ‘) (Jiang et al., 2007). Result and Discussion Amplification of the 16S rRNA gene was Marine Sediment-derived Actinomycetes performed in T100 thermal cycler (Bio-Rad) in Fourteen actinomycetes that isolated a total volume of 50 ml as a described above. from marine sediments in Krakal beach in PCR program used to follow Jiang et al. (2007) Indonesia and produce anticancer (Farida et with some modifi cations, the PCR program al., 2007) were used in this study. Based on included initial denaturation at 97°C for 3 min, colony morphology after 14 days of growth denaturation at 95°C for 1 min, then followed on solid medium starch nitrate at 30°C showed by annealing at 60°C for 1 min, extension at that all 14 actinomycetes diverse and different 72°C for 3 min the whole process lasted about from each other (Figure 1). From the colony 30 cycles, the last cycle followed by a final morphology also indicates that almost all extension at 72° C for 10 min and cooling stage isolates are most likely the genus Streptomyces.

Load more